Effects of UV-C radiation on extension growth, H+-extrusion and transmembrane electric potential in maize coleoptile segments

The effect of 253.7 nm ultraviolet radiation on elongation growth, medium acidification and changes in electric potential difference between vacuole and external medium in cells of maize ( Zea mays L.) coleoptile segments was investigated. It was found that irradiation with 390, 1170, 3900 and 5 850 J m⁻² UV-C (ultraviolet radiation 253.7 nm) inhibited elongation growth, whereas at 195 J m⁻² stimulation of growth was observed. The administration of IAA (10⁻⁵ M ) to the incubation medium of coleoptile segments partially abolished the inhibitory effect of UV-C. The pH of the incubation medium, measured simultaneously with growth, showed that the exposure of the segments to UV-C caused inhibition of H⁺-extrusion (or stimulation of H⁺ uptake). The presence of IAA (10⁻⁵ M ) in the incubation medium promoted (except after 5850 J m⁻² irradiation) H⁺-extrusion to a level comparable with that produced by IAA in non-irradiated segments. In UV-C irradiated segments the potential difference underwent significant alterations. Irradiation of coleoptile segments with 390 J m⁻² caused a transient depolarization, which was fully reversible within 30 min, while at higher doses depolarization was irreversible. The hyperpolarization of the membrane potential (MP) in cells of maize coleoptile induced by IAA was completely nullified by subsequent irradiation with UV-C. It is suggested that UV-C inhibited IAA-induced growth by a mechanism independent of cell wall acidification.     

Genetic analysis of a lactococcal plasmid replicon

Summary The sequence and genetic organization was determined of the 2508 by lactococcal portion of pFX2, which was derived from a crypticLactococcus lactis subsp.lactis plasmid and used as the basis for construction of a series of lactococcal vectors. A lactococcal plasmid plus origin and two replication protein-coding regions (repA andrepB) were located. RepA has a helix-turn-helix motif, a geometry typical of DNA-binding proteins. RepB shows a high degree of homology to the plasmid replication initiation proteins from other gram-positive bacteria andMycoplasma. The transcribed inverted repeat sequence betweenrepA andrepB could form an attenuator to regulate pFX2 replication. Upstream of theori site, and in a region which was non-essential for replication, a 215 by sequence identical to the staphylococcal plasmid pE194 and carrying the RS_A site was identified. The genetic organization of this lactococcal plasmid replicon shares significant similarity with pE194 group plasmids.     

The pH profile for acid-induced elongation of coleoptile and epicotyl sections is consistent with the acid-growth theory

The acid-growth theory predicts that a solution with a pH identical to that of the apoplast of auxintreated tissues (4.5–5.0) should induce elongation at a rate comparable to that of auxin. Different pH profiles for elongation have been obtained, however, depending on the type of pretreatment between harvest of the sections and the start of the pH-incubations. To determine the acid sensitivity under in vivo conditions, oat (Avena sativa L.) coleoptile, maize (Zea mays L.) coleoptile and pea (Pisum sativum L.) epicotyl sections were abraded so that exogenous buffers could penetrate the free space, and placed in buffered solutions of pH 3.5–6.5 without any preincubation. The extension, without auxin, was measured over the first 3 h. Experiments conducted in three laboratories produced similar results. For all three species, sections placed in buffer without pretreatment elongated at least threefold faster at pH 5.0 than at 6.0 or 6.5, and the rate elongation at pH 5.0 was comparable to that induced by auxin. Pretreatment of abraded sections with pH-6.5 buffer or distilled water adjusted to pH 6.5 or above gave similar results. We conclude that the pH present in the apoplast of auxin-treated coleoptile and stems is sufficiently low to account for the initial growth response to auxin.Abbreviations FS free space - IAA indole-3-acetic acid This research was supported by a grant from the National Adonautics and space Administration (NASA), NAGW 1394 to R.E.C., NASA grant NAGW-297 to M.L.E., and NASA grant NAG 1849 to D.L.R.     

Novel seed lipid phenotypes in combinations of mutants altered in fatty acid biosynthesis inArabidopsis

Summary The seed fatty acid (FA) composition of various single mutant combinations ofArabidopsis thaliana that affect FA biosynthesis has been examined. Double mutant combinations offae, a mutation affecting CIS elongation, and a series of four other FA biosynthetic mutants were synthesized. The four other single mutants were:fad2 andfad3, which are deficient in 181 and 182 desaturation, respectively;fab1, which is elevated in 160 and decreased in 181; andfab2, which is elevated in 180 and decreased in 181. The superimposition of two blocks in the FA biosynthetic pathway leads to dramatic changes in the FA content of the double mutants. The tenArabidopsis stocks analyzed to date (wild-type, five single mutants, and four double mutants) make seed oils with a wide range of FA compositions, and illustrate the diversity of oils it is possible to obtain from a single plant species.     

A nonconjugative mobilizable broad host range plasmid of Acinetobacter sp. that determines HgCl2 resistance

Summary HgCl₂-resistant strains of Acinetobacter sp. obtained from the soil at the Khaidarkan mercury mine (Kirghiz SSR) were found to contain, apart from large plasmids (60 kb), a small plasmid (7.5 kb) designated pKL1. It was established by conjugative crosses and transformation that pKL1 is a broad host range mobilizable plasmid and that it carries the Hg^r determinant. The restriction map of pKL1 was constructed; the site of the Hg^r determinant and the regions essential to replication were localized. A comparison of these results with earlier data suggests that microorganisms belonging to one microbiocenosis may carry Hg^r determinants on plasmids with highly different structures and properties.Deceased on July 16, 1985     

The frequency of mutants in human fibroblasts UV-irradiated at various times during S-phase suggests that genes for thioguanine- and diphtheria toxin-resistance are replicated early

Human cells deficient in rate of excision repair of DNA damage induced by UV-radiation, i.e., xeroderma pigmentosum (XP) cells, are much more sensitive to the mutagenic effect of UV than are cells from normal persons. The lower frequency of mutants in the latter cells has been attributed to the fact that, unlike XP cells, they excise most of the potentially mutagenic lesions before these can be converted into mutations. If semi-conservative DNA synthesis on a template still containing unexcised lesions is responsible for introducing mutations and if replication of the gene of interest, e.g., hypoxanthine (guanine)phosphoribosyltransferase (HPRT) for thioguanine resistance or the elongation factor 2 (EF-2) for diphtheria toxin resistance, occurs at a particular time during S-phase, it should be possible to shorten the time available for such repair by synchronizing cells and irradiating them just as the gene is to be replicated. The predicted result would be a much higher frequency of mutants at one part in the S-phase than at other times. To test this, cells were synchronized using the alpha-polymerase inhibitor aphidicolin, which blocks cells at the G1/S border. Autoradiography, cytofluorimetry, and incorporation of tritiated thymidine studies showed that DNA synthesis started immediately after release from aphidicolin and was completed in 8-10 h. Cells irradiated with 6 J/m2 at various times post-release were assayed for survival and mutations. The frequency of thioguanine- or diphtheria toxin-resistant cells in the population was highest in cells irradiated during the first fifth of the S-phase, i.e., 0-1.5 h post-release. It was significantly lower in cells irradiated at later times. In contrast, UV-induced cytotoxicity showed no significant time dependence during S-phase. These data suggest that the HPRT and EF-2 genes are replicated early in S-phase.     

Acetic acid esters and permeable weak acids induce active proton extrusion and extension growth of coleoptile segments by lowering the cytoplasmic pH

In Avena coleoptile segments a decrease of cytoplasmic pH activates energy-dependent H⁺ extrusion into the apoplast, thereby triggering extension growth. This sequence of events cannot be inhibited by cycloheximide and is induced by the following conditions and compounds. (i) A short anaerobic treatment of coleoptile segments results in the formation of lactic acid and an intracellular decrease of pH. For a period of 20 min after transfer to normal air, the growth rate is up to six times higher than the rate before anaerobiosis. (ii) Similarly, incubation of segments with CN^– (0.1 mM) in the presence of oxygen causes and accumulation of lactic acid and a fall in cell-sap pH. After removing CN^– a growth burst occurs. (iii) Higher concentrations of permeable acids (10 mM in buffer pH 5.8) induce extension growth. This growth is O₂-dependent and therefore differs from the acid growth, which can be triggered under anaerobic conditions by acid buffers of pH5 via the direct increase of cell-wall plasticity. (iv) A short application of CO₂-saturated buffer (pH 5.8) causes CO₂-induced elongation growth; after a 3-min pulse the growth rate is enhanced for about 15 min. (v) Lipophilic esters of acetic acid or propionic acid, such as naphthylacetate, naphthylpropionate, phenylacetate, benzylacetate induce elongation growth. These compounds, when taken up into the cell, are hydrolized by esterases; the acids released lower the cytoplasmic pH (shown by the pH indicator, fluorescein). The highest esterase activity was found in a microsomal membrane fraction of coleoptiles. While the carboxyester-induced extension growth is completely inhibited under anoxia, the initial acidification of the bathing solution can still be observed. This decrease in external pH is obviously the result of ester hydrolysis, caused by damaged cells, and is not the result of pH changes within the cell-wall compartment. It is suggested that a fast uptake of carboxyesters and the shift in equilibrium caused by their internal hydrolysis leads to a continuous formation of acids which lowers the cytoplasmic pH and activates the ATP-dependent H⁺ extrusion. In most experiments fusicoccin (a diacetic acid ester) acts similarly to naphthylacetate and the other carboxyesters, although quantitative differences exist. Therefore, it is possible that fusicoccin is effective partly on the basis of its ester characteristic. The effects observed are discussed with regard to the very narrow pH optimum of plasma-membrane H⁺-ATPases exhibiting their highest levels of activity at pH 6.5 (Hager and Biber 1984, Z. Naturforsch. C 39, 927–937).Abbreviations CHM cycloheximide - DMO dimethadione (5.5-dimethyl-2,4-oxazolidinedione) - FC fusicoccin - IAA indole-3-acetic acid - Mes 2-(N-morpholino)ethanesulfonic acid - NA (or )-naphthylacetate (acetic acid-1(or-2-)naphthylester) - NAA (or )-naphthaleneacetic acid - PA phenylacetate (acetic acid phenylester)     

Effect of applied and endogenous indol-3-yl-acetic acid on maize root growth

The level of endogenous Indol-3-yl-acetic acid (IAA) measured by gas chromatography-mass spectrometry in the elongating zone of intact primary roots of Zea mays showed a good linear correlation with the growth rate of these roots. When they were treated with IAA, their relative elongation decreased; this indicates a supraoptimal content of endogenous IAA. However, the growth of some of the relatively rapidly extending roots was enhanced by such treatment. Interactions between endogenous and applied IAA in the control of root growth are discussed.Abbreviations GC-MS gas chromatography-mass spectrometry - IAA Indol-3-yl-acetic acid     

Control of the rate of cell enlargement: Excision,wall relaxation,and growth-induced water potentials

A new guillotine thermocouple psychrometer was used to make continuous measurements of water potential before and after the excision of elongating and mature regions of darkgrown soybean (Glycine max L. Merr.) stems. Transpiration could not occur, but growth took place during the measurement if the tissue was intact. Tests showed that the instrument measured the average water potential of the sampled tissue and responded rapidly to changes in water potential. By measuring tissue osmotic potential ( _s ), turgor pressure ( _p ) could be calculated. In the intact plant, _s and _p were essentially constant for the entire 22 h measurement, but _s was lower and _p higher in the elongating region than in the mature region. This caused the water potential in the elongating region to be lower than in the mature region. The mature tissue equilibrated with the water potential of the xylem. Therefore, the difference in water potential between mature and elongating tissue represented a difference between the xylem and the elongating region, reflecting a water potential gradient from the xylem to the epidermis that was involved in supplying water for elongation. When mature tissue was excised with the guillotine, _s and _p did not change. However, when elongating tissue was excised, water was absorbed from the xylem, whose water potential decreased. This collapsed the gradient and prevented further water uptake. Tissue _p then decreased rapidly (5 min) by about 0.1 MPa in the elongating tissue. The _p decreased because the cell walls relaxed as extension, caused by _p , continued briefly without water uptake. The _p decreased until the minimum for wall extension (Y) was reached, whereupon elongation ceased. This was followed by a slow further decrease in Y but no additional elongation. In elongating tissue excised with mature tissue attached, there was almost no effect on water potential or _p for several hours. Nevertheless, growth was reduced immediately and continued at a decreasing rate. In this case, the mature tissue supplied water to the elongating tissue and the cell walls did not relax. Based on these measurements, a theory is presented for simultaneously evaluating the effects of water supply and water demand associated with growth. Because wall relaxation measured with the psychrometer provided a new method for determining Y and wall extensibility, all the factors required by the theory could be evaluated for the first time in a single sample. The analysis showed that water uptake and wall extension co-limited elongation in soybean stems under our conditions. This co-limitation explains why elongation responded immediately to a decrease in the water potential of the xylem and why excision with attached mature tissue caused an immediate decrease in growth rate without an immediate change in _p Abbreviations and symbols L tissue conductance for water - m wall extensibility - Y average yield threshold (MPa) - _o water potential of the xylem - _p turgor pressure - _s osmotic potential - _w water potential of the elon gating tissue